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 Table of Contents  
Year : 2023  |  Volume : 8  |  Issue : 2  |  Page : 173-180

Evaluation of the anti-inflammatory activity of hexane and ethanolic extracts of polyherbal formulation of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) on carrageenan-induced paw edema in wistar rats

1 Department of Pharmacy, Rajiv Gandhi Proudyogiki Vishwavidhyalay, Bhopal, India
2 Department of Zoology, Sri Satya Sai University of Technology and Medical Sciences, Sehore, Madhya Pradesh, India

Date of Submission07-Jun-2022
Date of Acceptance21-Nov-2022
Date of Web Publication31-Mar-2023

Correspondence Address:
Abu Tahir
Department of Pharmacy, Rajiv Gandhi Proudyogiki Vishwavidhyalay, Bhopal 462033, Madhya Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jdras.jdras_85_22

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BACKGROUND: Inflammation is a primary physiological defence mechanism that assists the body in protecting itself from infection, toxic chemicals, or other noxious stimuli. The current study demonstrated phytochemical screening, anti-inflammatory activities, and sub-acute toxicity of hexane and ethanol extracts of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (Fruits), as well as anti-oxidant activity. The Carrageenan-Induced Rat Paw Edema method was used to assess anti-inflammatory activity. METHODS: The carrageenan-induced paw edema test evaluated anti-inflammatory activity. Male albino Wistar rats weighing 150 ± 10g were divided into six groups of six animals each. Paw edema was induced with 1.5% carrageenan in all the groups except the normal. Group, I received a plain control of 1 ml of 1% Carboxy Methyl Cellulose (CMC); Group II standard drug received Indomethacin (10 mg/kg); Group III received Polyherbal formulation of hexane extracts (PHFH) 250 mg/kg b.w; Group IV received PHFH 500 mg/kg b.w.; Group V was given 250 mg/kg b.w. of Polyherbal formulation of ethanolic extracts (PHFE). The extract’s acute toxicity (2000 mg/kg) as per OECD guidelines was studied in albino rats for 14 days. The qualitative analysis of various phytochemical constituents of various phytoconstituents was determined. The DPPH method was used to evaluate anti-oxidant activity. RESULTS: The results showed that both PHFH and PHFE exhibited marked inhibition of the edema size from 1, 3, and 5 hrs of study as compared to the standard drug indomethacin (10 mg/kg b.w). The PHFE (250 and 500 mg/kg) displayed excellent protection against inflammation to PHFH (250 and 500 mg/kg). Compared to the standard drug, indomethacin which showed the highest excellent protection against inflammation. PHFE has lower anti-oxidant activity than standard ascorbic acid but exhibits higher anti-oxidant activity than PHFH. In an acute toxicity test, hexane-ethanolic extracts up to 3000 mg/kg had no toxic effects. CONCLUSION: From this study, we conclude that Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) have anti-inflammatory activity by reducing paw inflammation as well as showing anti-oxidant activity.

Keywords: Anti-inflammatory, antioxidant, iper longum, nigella sativa, ocimum tenuiflorum, polyherbal formulation

How to cite this article:
Tahir A, Dar MS. Evaluation of the anti-inflammatory activity of hexane and ethanolic extracts of polyherbal formulation of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) on carrageenan-induced paw edema in wistar rats. J Drug Res Ayurvedic Sci 2023;8:173-80

How to cite this URL:
Tahir A, Dar MS. Evaluation of the anti-inflammatory activity of hexane and ethanolic extracts of polyherbal formulation of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) on carrageenan-induced paw edema in wistar rats. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Jun 10];8:173-80. Available from: http://www.jdrasccras.com/text.asp?2023/8/2/173/373021

  Introduction Top

The term ‘inflammation’ comes from the Latin word ‘inflammo,’ which means ‘to set alight; to ignite, or to set on fire.’ Inflammation is referred to as a hot thing in Greek. Within, the Greek word phlegmon has been used to define inflammatory lesions.[1],[2] Inflammation has always been a mystery to humans. This phenomenon can be triggered by something as minor as a bruise or as serious as a myocardial infarction. Non-steroidal anti-inflammatory drugs (NSAIDS) and corticosteroids are the most commonly used anti-inflammatory drugs, but their toxic side effects include epigastric distress, peptic ulceration, osteoporosis, and iatrogenic Cushing’s syndrome limited their use.[3],[4] It is a normal reaction to disruptions in homeostasis caused by infection, injury, or trauma.[2],[5],[6],[7] Waram (inflammation) is a broad term in Unani medicine that refers to any abnormal swelling caused by the accumulation of blood, pus, water, or flatus.[8] A swelling known as a waram is brought on by an abnormal substances absorbing into an organ.[9],[10]

According to the WHO, 70–80% of the world’s population relies on non conventional medicine, primarily from herbal sources, for primary health care.[11],[12] Its popularity is growing, particularly in developing countries where the cost of consulting a physician and the cost of medicine are out of reach for the majority of people.[13]These anti-inflammatory medications are used to relieve pain in various conditions such as arthritis, muscle pain, and ligament pain. Conventional drug treatments are not very exclusive in controlling the occurrence and outcome of many inflammatory diseases. In addition, they cause significant side effects in patients.[14]

Herbal medicines derived from plant extracts are increasingly being used to treat a wide range of clinical diseases, despite little being known about their mode of action. The pharmacological evaluation of various plants used in Indian traditional systems of medicine is gaining popularity. It is known that Ayurvedic herbals are prepared in many dosage forms, mostly of which are PHF (Polyherbal formation). In the current scenario, Plant-derived medicinal compounds such as flavonoids, saponins, alkaloids, terpenoids, glycosides, and coumarins could provide an excellent starting point for developing new anti-inflammatory agents that are more efficacious, safer, more affordable, and more accessible to patients.

The plant has been linked to various pharmacological activities, including diuretics, CNS depressants, diaphoretic and styptic properties. Plant decoction has activity in renal and urinary complications. Plant seeds have anti-infammatory activity, and plant roots are used to treat cancer and scrofulous tumours.[15],[16]Carrageen-induced rat paw edema exhibits numerous biochemical and cellular characteristics that have been clearly identified in the background and are constantly being revised in light of discoveries.[17],[18]

Based on the nature of the interaction, there are two mechanisms for how synergism acts (i.e., pharmacodynamics and pharmacokinetic). In terms of pharmacokinetic synergism, the ability of the herb to facilitate the absorption, distribution, metabolism and elimination of other herbs is focused. On the other hand, pharmacodynamic synergism studies the synergistic effect when active constituents with similar therapeutic activity are targeted to a similar receptor or physiological system. Other than that, it is believed that multiple factors and complications cause diseases in most cases, leading to visible and invisible symptoms. Here, the combination of herbals may act on multiple targets at the same time to provide thorough relief.[19]

Due to synergism, polyherbalism confers some benefits unavailable in a single herbal formulation. Better therapeutic effect can be reached with a single multi-constituent formulation. For this, a lower dose of the herbal preparation would be needed to achieve desirable pharmacological action, thus reducing the risk of deleterious side effects. Besides, PHFs improve patients’ convenience by eliminating the need to take more than one different single herbal formulation at a time, which indirectly leads to better compliance and therapeutic effect. All these benefits have resulted in the popularity of PHF in the market when compared to single herbal formulation.[20]This study is aimed to evaluate anti-oxidant and anti-inflammatory effect of PHF prepared by the combination of Nigella sativa (seeds), Ocimum sanctum L. (leaves) and Piper longum L. (fruits) hexane and ethanol extracts individually in different doses on carrageenan-induced rat paw edema model. This approach is an alternative method to discover potentially new herbal combinations/formulations for the treatment of inflammation and its symptoms.

  Material and Methods Top

Plant materials and authentication

Plant materials of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) were collected locally from Bhopal, M.P. And the authentication of all the three plants were done by Dr. Zia Ul Hasan, Department of Botany, Safia College of Science, Peergate, Bhopal, M.P India. A voucher specimen of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) were deposited for record with the specimen number 332/Bot/Safia/18, 366/Bot/Safia/18 and 418/Bot/Safia/18, respectively.

Extraction and preparation of hexane and ethanolic extracts of each selected plant


The seeds of Nigella sativa L., leaves of Ocimum tenuiflorum L., and fruits of Piper longum L. were air-dried up to 7 days. Dried plant parts were stored in air-tight glass containers in dry and cool place to avoid contamination and deterioration.[21] Each air-dried medicinal plant (100 gm) was coarsely powdered and extracted by cold extraction method (double maceration) with hexane and ethanol at 25°C for 48 hours. The extracts were individually filtered through laboratory filter paper, and the filtered solutions were dried using a rotary evaporator under reduced pressure (335 mbar) at 40 ± 2°C and stored in the refrigerator (2–4°C) for further activity.

Preparation of polyherbal formulations (suspension)

Dried hexane extract of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) were mixed in 1:1:1 ratio and suspended in double distilled water with carboxymethyl cellulose sodium (CMCS) (2%) to prepare polyherbal formulation of hexane extracts (PHFH). Similarly, dried ethanolic extractof Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (Fruits) were mixed in 1:1:1 ratio and suspended in double distilled water with carboxymethyl cellulose sodium (CMCS) (2%) to prepare polyherbal formulation of ethanolic extracts (PHFE).[22]

Preliminary phytochemical analysis

The phytochemical anaylsis of hexane and ethanol extracts of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) was performed in 100 ml of its own mother solvents to obtain a stock with a concentration of 1% (v/v). The resulting extracts were subjected to preliminary phytochemical screening using standard phytochemical screening methods[23],[24]

Experimental animals

Wistar rats (150 ± 10 g) were housed in groups of six (n = 6) under a standard 12 h light/dark cycle and temperature and humidity control (25 ± 2°C, 55–65%). Rats were fed standard rodent chow and given unlimited access to water. Before the experiments, rats were acclimatised to laboratory conditions for 7 days. The experimental work was conducted at Pinnacle Biomedical Research Institute (PBRI), Bhopal, India. With protocol approval reference number was PBRI/IAEC/PN-18010. The animal studies were approved by the Institutional Animal Ethics Committee (IAEC), which was established by the Ministry of Environment and Forests, Government of India, for the purpose of controlling and supervising experimental animals (CPCSEA Reg No.-1824/PO/ERe/S/15/CPCSEA).

Acute oral toxicity

Acute toxicity testing of plant materials prepared extracts was performed following the Organization for Economic Cooperation and Development (OECD) Guidelines-423.[25] The animals were fasted for four hours but had free access to water the entire time. According to OECD recommendations, the starting dose level should be that which is most likely to cause mortality in some of the dosed animals; and when no information is available on a substance to be tested in this regard; for animal welfare reasons, the dose level to be used as the starting dose is chosen from one of three fixed levels of 50, 100, 300, and 2000 mg/kg body weight. The selected animals were alienated into 4 different groups. All animals were kept as per conditions mentioned in the guidelines. The dose selected for both (PHFH, PHFE) was 2000 mg/Kg. The variations in consciousness, abnormalities, rate of respiration, skin colour, behaviour, excretion, impairment in diet intake, water feeding, and loss of hair or death of test animals were observed first 5 h, 12 h and every day for 14 days.[26]

In vitro anti-oxidant assay

DPPH assay method

2 ml solution of both PHFH and PHFE in different concentrations (20 μg/ml-100 μg/ml) were added individually to 2 ml of DPPH solution (75 μM). Standard solution was ascorbic acid in the same concentrations as samples. The reaction mixtures were shaken thoroughly and kept in the dark for 30 minutes. The solution was prepared by adding methanol (2 ml) to DPPH solution (2 ml).[27] The absorbance of all the samples and control solution was measured at 517nm using UV-Visible spectrophotometer and the remaining DPPH was calculated. The radical scavenging activity was expressed as the percentage inhibition and was calculated using the following formula:

Percentage of Inhibition = [(AC - AS)/AC] X 100.

Where AC is the absorbance of the control and AS is the absorbance of the Sample. The graphs were plotted between % inhibition and different concentrations of PHFH, PHFE and standard ascorbic acid individually and IC50 value was determined.

Chemicals and drugs

Indomethacin (purchased from a local pharmacy store), Carrageenan (Sigma Aldrich, India), All of the chemicals used in this experiment were of the analytical grade.

Design of Experiment

Six groups of six animals were formed at random from the animals. Group I control group receives Carrageenan 1% (sub-plantar injection) + 0.5 ml of Carboxymethyl cellulose sodium (CMCS) (2%), orally. Group II Standard group receives Carrageenan 1% (sub-plantar injection) + Indomethacin (10 mg/kg b.w), orally. Group III and IV Treatment groups receives Carrageenan 1% (sub-plantar injection) + PHF prepare by hexane extract (250 mg/kg and 500 mg/kg), orally. Group V and VI Treatment groups receives Carrageenan 1% (sub-plantar injection) + PHF prepare by ethanolic extract (250 mg/kg and 500 mg/kg), orally respectively.

Anti-inflammatory activity (in-vivo)

Carrageenan-induced paw edema (CIPE)model

Evaluation of anti-inflammatory activity of both PHFH and PHFE was performed on male Wistar albino rats (150 ± 10g) by CIPE model. Animals of all groups were administered with 0.1 ml sub-plantar injection of carrageenan (1%) in normal saline in the left hind paw to induce acute inflammation, 1 h after oral drug treatment.[28] The volume of paw of each rat was measured by digital plethysmometer at 1,3 and 5 h after administration of carrageenan injection.[29]The variation between initial and the successive readings represented the rate of inflammation or edema volume and the percent inhibition were measured.[22]

The following equation was used to calculate the percent edema inhibition:

Where, Vc represented the mean increase in paw volume of control group, and Vt represented the mean increase in paw volume of test and standard drugs. The results were presented as SD

Statistical analysis

All values were expressed as mean ± SD (n = 6 in each group). One-way ANOVA was applied to test for the significance of biochemical data of the different groups. P-values less than 0.05 were regarded as statistically significant.

  Results Top

Results of preliminary screening of phytochemicals and in vitro anti-oxidant assay by DPPH method along with anti-inflammatory activity by carrageenan-induced paw edema method were analyzed statistically and presented in [Table 1][Table 2][Table 3][Table 4][Table 5] and [Figure 1][Figure 2][Figure 3][Figure 4][Figure 5].
Table 1: Anti-inflammatory activity of polyherbal formulations (PHFH and PHFE)

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Table 2: Qualitative phytochemical analysis of ethanolic extracts

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Table 3: Qualitative phytochemical analysis of hexane extracts

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Table 4: DPPH scavenging activities of extracts of PHFH, PHFE andAscorbic acid

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Table 5: Percentage inhibition in anti-inflammatory activity of polyherbal formulations (PHFH and PHFE)

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Figure 1: DPPH assay by using standard ascorbic acid

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Figure 2: DPPH assay by using PHFH

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Figure 3: DPPH assay by using PHFE

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Figure 4: Paw volume at different hours 1, 3 and 5 of PHFH and PHFE at 250 mg/kg and 500 mg/kg

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Figure 5: Percentage inhibition of PHFH and PHFE at 250 mg/kg and 500 mg/kg

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  Discussion Top

The carrageenan-induced rat paw edema model is an appropriate test for evaluating anti-inflammatory drugs, and it has frequently been used to assess the drug’s anti-edematous effect. Carrageenan is a powerful chemical that stimulates the release of inflammatory and pro-inflammatory mediators (prostaglandins, leukotrienes, histamine, bradykinin, TNFα, and so on).[30],[31]

Acute or chronic inflammation is the two types of inflammation. Acute inflammation is the body’s initial response to injurious stimuli, manifested by increased movement of plasma and leukocytes from the blood into the injured tissues. Acute inflammation is initiated by cells already present in the tissues. This is characterized by significant vascular changes, such as vasodilatation and increased capillary permeability, caused by the actions of various inflammatory mediators. Chronic inflammation is a long-term inflammatory response that results in a progressive shift in the type of cells present at the site of inflammation and is characterized by the inflammatory process’s simultaneous destruction and healing of tissues.

The response to carrageenan-induced paw edema is biphasic.[32],[33],[34]

The first phase was mediated by the release of histamine, serotonin, and kinin, whereas the second phase was mediated by the release of prostaglandins and slow-acting substances, which peaked at 4 hours. According to reports, the second phase is sensitive to both steroidal and non-steroidal anti-inflammatory agents.[35] Prostaglandins are the primary cause of acute inflammation. NSAIDs, in general, strongly inhibit the second phase of carrageenan-induced edema, whereas others inhibit both phases. The ability of a compound to reduce local edema induced in the rat paw by injection of an irritant agent is the most widely used primary test for screening new anti-inflammatory agents. N. sativa (seeds), O. sanctum L. (leaves) and P. longum (fruits) may contain anti-inflammatory agents that are responsible for the inhibition of prostaglandins and the inflammatory pathway. Anti-inflammatory activity of polyherbal formulations was evaluated by carrageenan-induced rat paw edema method and outcomes are given in [Table 1].

The anti-inflammatory activity of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) hexane and ethanol extracts may be due to a combination of different biological constituents rather than any single compound. The presence of secondary metabolites such as alkaloids, flavonoids, glycosides, phenols, saponins, sterols, and others may contribute to the curative properties of medicinal plants. [Table 2] and [Table 3] show the presence of flavonoids, glycosides, phenols, fatty acids, steroids, and terpenoids in successive extracts of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits).

Under acute toxicity test, oral administration of both the polyherbal formulations (PHFH, PHFE) at the dose of 2000 mg/ Kg body weight exhibited no evidence of any abnormalities, significant behavioral changes, skin sensitivity or deaths. This may be due to the fact that all the three plants Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (Fruits) have been traditionally used for many years by humans to treat various disease ailments without any reported incidence of adverse effects.

[Figure 1][Figure 2][Figure 3] showed the anti-oxidant activity of the polyherbalformulations compared with ascorbic acid. IC50 values of ascorbic acid, PHFH and PHFE were 13.725μg/ml, 74.27μg/ml and 47.74μg/ml, respectively. According to the findings, PHFE has lower anti-oxidant activity than ascorbic acid but higher anti-oxidant potential than PHFH [Table 4]. The scavenging effect of polyherbal formulations(PHFH, PHFE) on DPPH might be due to the hydrogen contributing abilityof active constituents present in plant extracts.[36] Though, the scavenging potential of both the PHFs were found less than that of ascorbic acid,the results supported the proton-donating potential of both the PHFs as primary anti-oxidants.

The results showed that both PHFH and PHFE were exhibited appreciable inhibition of the edema size from 1, 3, and 5 hrs of study as compared to the standard drug indomethacin [Figure 4]. The PHFE emerged as the most promising formulation in doses of 250 and 500mg/kg [Figure 5], also displayed excellent protection against inflammation with percent protection of 43.24% and 56.20%, respectively [Table 5]. The PHFH also showed considerable inhibition of the edema size in both doses of 250 and 500mg/kg [Figure 5], also exhibited protection against inflammation with percent protection of 25.6%and36.0%, respectively [Table 5]. However, the standard indomethacin (10 mg/kg) showed the highest inhibition of edema size and showed remarkable protection against inflammation with percent protection of 81.40% protection [Table 5].

On the basis of above outcomes, it can be inferred that the PHFE inhibitory effect against inflammation induced by carrageenan is more potent when compared to PHFH. It is postulated that the inflammatory potential of both the polyherbal formulations (PHFE and PHFH) may be due to the COX enzyme inhibition potential of constituents present in plant extracts.[37] The study’s findings revealed that various phytochemicals are present in plant extracts and may be responsible for pharmacological activity. Plant extracts reduces pain in rats, leading to the conclusion that it has potent anti-infammatory activity.

  Conclusion Top

The antiinflammatory properties of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) were observed in present study. It is also proposed that the mechanisms of action of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits) are related to the inhibition of histamine, serotonin, and prostaglandin synthesis. However, more research is needed to isolate and characterise anti-inflammatory chemical constituents found in hexane and ethanolic extracts of Nigella sativa L. (seeds), Ocimum tenuiflorum L. (leaves), and Piper longum L. (fruits).


The authors would like to express their gratitude to Dr. Megha Jha,Sr. Scientific Manager (RandD)of PBRI, for providing the best possible facilities for the smooth conduct of this research study.

Financial support and sponsorship


Conflicts of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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